Staphylococcal nuclease is a small globular protein without any disulfide bonds. It has been used extensively for protein folding studies. The NMR spectrum of this protein contains several additional resonances due to protein conformational heterogeneities. Replacement of Pro-47 and Pro-117 with glycines eliminates these heterogeneities in the NMR spectrum. Time-resolved fluorescence studies suggest that these mutations cause a decrease in the protein chain mobility. In order to further examine the effect of these mutations, the crystal structure of the Pro-47 to Gly and Pro-117 to Gly double mutant has been solved. Two additional hydrogen bonds between Tyr-115 and other residues are present in this structure and not in the WT structure. Future studies will include a comparison of the hydrogen exchange behavior in WT nuclease and in the Pro to Gly mutants.